ES2960550T3 - Quantification of nucleosome modifications using chemically defined recombinant nucleosomes - Google Patents

Abstract

The invention relates to the use of recombinant/semi-synthetic nucleosomes carrying histone and/or DNA modifications as a reference standard for the quantification of covalently modified (on histone proteins or wrapping DNA), variant or mutant nucleosomes (collectively "modified nucleosomes" or "nucleosome modifications") from a biological sample. The invention further relates to methods for using the assay to accurately quantify single or combinatorial nucleosome modifications as disease biomarkers. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Quantification of nucleosome modifications using chemically defined recombinant nucleosomes

Priority statement

The present application claims the benefit of the United States provisional application with serial number 62/637,066, <filed on>March 1, 2018.>

field of invention

The invention relates to the use of recombinant/semi-synthetic nucleosomes carrying histone and/or DNA modifications as a reference standard for the quantification of covalently modified nucleosomes (in histone proteins or envelope DNA), variants or mutants (collectively " modified nucleosomes") or "nucleosome modifications") from a biological sample. The invention further relates to methods for using the assay to accurately quantify individual or combinatorial nucleosome modifications as biomarkers of disease.

Background of the invention

Nucleosomes are the repeating units of chromatin, consisting of 147 base pairs of DNA that integrally surround a histone octamer (containing >2 copies of each of the core histones) (H2A, H2B, H3 and H4)) (Margueron et al., Nat. Rev. Genet. 11 (4):285 (2010)). Changes in chromatin structure and function regulate various cellular activities, including gene expression, DNA repair, chromosome transmission, and cell differentiation (Brown et al., Hum. Mol. Genet. 21(R1) :R90 (2012)); Lahtz et al., J. Mol. Cell. Biol. 3(1):51 (2011); Lunyak et al., Hum. Mol. Genet. 17(R1):R28 (2008); Reik, Nature 447(7143):425 (2007)). These processes are mediated in part by reversible histone posttranslational modifications (PTMs; e.g., lysine methylation or acetylation), which have direct effects or are “read” by effector binding proteins to transduce specific downstream signaling pathways. To date, more than 100 individual histone PTMs have been identified, many of which are related to human diseases, ranging from neurodegeneration (Landgrave-Gomez et al., Front. Cell. Neurosci. 9:58 (2015) ) and metabolic syndrome (DelCurto et al., Curr. Opin. Clin. Nutr. Metab. Care 16(4):385 (2013); Wang et al., Antioxid. Redox Signal 17(2):282 (2012) ) to cancer (Chopra et al., Cancer Genet. 208(5):192 (2015); Greenblatt et al., Leukemia 28(7):1396 (2014); Gajer et al., Oncogenesis 4:e137 (2015) ;

Witt et al., Curr. Pharm. Des. 15(4):436 (2009); Hanmod et al., Pediatr. Blood Cancer 62(1):52 (2015); Kobayashi et al., Oncogene 32(21):2640 (2013)). Significantly, PTM-interacting proteins (also known as 'readers') are highly amenable to drug modulation, making them exceptional therapeutic targets for numerous human diseases and disorders (Arrowsmith et al., Nat. Rev. Drug Discov .

11 (5):384 (2012)). Furthermore, histone PTMs are an emerging class of cancer biomarkers that may be useful for early detection and prognosis of diseases, as well as informing personalized treatment strategies (Khan et al., World J. Biol. Chem. 6(4):333 (2015)); Chervona et al., Am. J. Cancer Res.

2(5):589 (2012)).

Cooperative function between histone PTMs and chromatin regulatory proteins represents a complex systems-level signaling network, and its effective interrogation requires nucleosome-based tools. Multivalency (i.e., multiple reader domains within a given protein) is a common feature of chromatin regulators (Ruthenburg et al., Nat. Rev. Mol. Cell. Biol. 8(12):983 (2007)) , and is believed to serve as a key means by which histone interactions are regulated in a context-specific manner. Indeed, reader binding (and subsequent changes in cellular regulation) is greatly altered in the presence of several PTMs or combinations thereof, a hypothesis known as the histone code (Strahl et al., Nature 403(6765) :45 (2000); Jenuwein et al., Science 293(5532):1074 (2001)). For example, the PHD bromodomain finger transcription factor (BPTF) reader protein contains domains with low individual binding affinity for H3K4me3 (Kd = ~1 pM) and H4K12ac (Kd = ~60 pM) (Li et al., Nature 442(7098):91 (2006)), but this is significantly increased (>3-fold versus H3K4me3 alone) in the presence of H3K4me3/H4K12ac combinatorially modified mononucleosomes (Ruthenburg et al., Cell 145(5):692 (2011) ). BPTF is a target for anticancer therapy (Dar et al., J. Natl. Cancer Inst. 107(5) (2015)); with elevated levels that is also found in patients with amyotrophic lateral sclerosis (Mu et al., Exp. Neurol. 146(1):17 (1997)). Interestingly, H3K4me3 and H3/H4 acetylation coexist at active promoters (Zhang et al., EMBO Rep. 16(11):1467 (2015)), and H3K4me3 has been shown to promote histone acetylation by recruiting of effectors (Tang et al., Cell 154(2):297 (2013)). Mechanistic studies have only begun to unravel the combinatorial effect of histone PTMs on cellular physiology, a necessary precursor to investigating the disease state. For example, changes in the gut microbiome (triggered by diet) can have a significant effect on specific combinations of histone PTMs in the host tissue (Krautkramer et al., Mol. Cell 64(5):982 (2016)) . Similarly, recent findings suggest that combinatorial nucleosome PTMs (versus individual PTMs) may provide better prognostic biomarkers for lung cancer (Shema et al., Science 352(6286):717 (2016)).

Efficient presentation of combinatorial histone PTMs depends on the three-dimensional structure of the intact nucleosome, which serves as a scaffold for physiological PTM-protein interactions. Understanding the nature of the combinatorial histone code is vital if we are to translate the connection between epigenetic regulation and disease into next-generation therapeutics and biomarkers. This is particularly true for histone methylation and acetylation, which play an integral role in chromatin regulation and disease (Greer et al., Nat. Rev. Genet. 13(5):343 (2012)); Filippakopoulos et al., Nat. Rev. DrugDiscov. 13(5):337 (2014); Soshnev et al., Mol. Cell 62(5):681 (2016)).

Several procedures have been developed for the quantification of histone PTMs from biological samples (Sidoli et al., J. Vis. Exp. 2016(111); Onder et al., Expert Rev. Proteomics 12(5):499 ( 2015); most of which depend on the use of modification-specific antibodies for nucleosome enrichment (e.g. immunoprecipitation). chromatin (Chromatin ImmunoPrecipitation); ChIP) or detection (for example, ELISA or Alfa). ELISA is commonly used to quantify histone or DNA modification from biological samples, with specific assays developed to directly quantify histone or nucleosome modifications using various antibody capture approaches. Nucleosome-based detection outperforms histone-based detection for two reasons:

1<) nucleosome-based procedures do not require acid extraction steps, which are laborious>and introduce variability; and,

2<) Nucleosome-based assays allow the quantification of combinatorial modifications in trans (for example, histone-histone, DNA-DNA or histone-DNA combinations), which are impossible to monitor when using histone subunits or DNA alone.

WO 2015/117145 discloses a method for quantitatively measuring the density of a first epitope of a core histone at a genomic locus in chromatin of a cell of a patient, wherein the first epitope of the core histone comprises at least one modification post-translational amino acid. WO 2005/019826 discloses the use of cell-derived nucleosomes from chicken cells as assay standards. WO 2015/104431 discloses biochemical assays that use recombinant modified nucleosomes as substrates to measure protein binding interactors or enzymatic activity.

Despite these important advantages, current nucleosome-based assays lack adequate controls for quantification. Indeed, assays using the current technique are qualitative, with PTM levels reported as relative measurements or normalized using exogenous (e.g., bird, yeast, or insect) xenochromatin. The use of purified exogenous chromatin preparations can present a variety of problems, as these reagents are poorly defined (at the specific PTM level) and show batch variability, which limits their use for assay standardization (between experiments or even laboratories). In addition to this, it is currently challenging to determine whether combinatorial marks actually occur on the same nucleosome rather than coinciding in the total cluster.

Given the above, there is a need in the art for improved controls for nucleosome-based assays that detect histone and/or DNA modifications from biological samples.

Summary of the invention

The present invention relates to the use of recombinant/semi-synthetic/designer nucleosomes (hereinafter referred to as "recombinant nucleosomes") carrying histone and/or DNA modifications as quantification standards for calibration. Unlike the purified chromatin extracts currently used, the present assay uses fully defined recombinant/semi-synthetic nucleosomes for precise quantification of individual or combinatorial nucleosome modifications. Similar to standards commonly used in the art, the modified nucleosomes will be assayed in the same experiment (treated identically to the samples) to generate a standard curve for assay quantification. Quantification of individual or combinatorial nucleosome PTMs may provide better disease biomarkers than the relative measurements currently in use.

Significantly, the inventors have determined that only nucleosomes provide useful reference standards in plasma samples. When histones or nucleosomes are added to plasma, only the latter are recovered to the predicted values and are therefore viable for quantification. In this way, the nucleosomes of the present invention provide useful standards for assays that quantify circulating nucleosomes directly from plasma or other body fluids.

The invention is defined by the appended claims included herewith. In addition to the invention, one aspect of the present disclosure relates to a method for quantifying the abundance of a nucleosome modification (histone or DNA) in a biological sample, the method comprising:

to. isolate a biological sample;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at a target epitope;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising a core histone and/or DNA modification target epitope;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding an affinity reagent to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard; and

g. Quantify the abundance of nucleosome modification at the target epitope by comparing the relative abundance in the native nucleosome library with the reference standard.

Another aspect of the present disclosure relates to a method for quantifying the abundance of two or more modifications (histone or DNA) in an individual nucleosome in a biological sample, the method comprising:

to. isolate a biological sample;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising two or more core histone and/or DNA modifications at a target epitope;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying two or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding two or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard; and

g. quantify the abundance of nucleosome modification on target epitopes by comparing the relative abundance in the native nucleosome library with the reference standard.

A further aspect of the present disclosure relates to a method for quantifying the abundance of one or more nucleosome modifications (histone or DNA) in a biological sample from a subject suffering from a disease or disorder, the method comprising:

to. isolate a biological sample from the subject;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at one or more target epitopes;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying one or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding one or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard; and g. Quantify the abundance of nucleosome modification at the target epitope by comparing the relative abundance in the native nucleosome library with the reference standard.

A further aspect of the present disclosure relates to a method for determining a prognosis for a subject suffering from a disease or disorder associated with epigenetic modifications based on the quantification of one or more nucleosome modifications, the method comprising:

to. isolate a biological sample from the subject;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at one or more target epitopes;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying one or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding one or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard;

g. quantify the abundance of nucleosome modification on target epitopes by comparing the relative abundance in the native nucleosome library with the reference standard; and

h. determine the prognosis of the subject based on the abundance of one or more target epitopes.

Another aspect of the present disclosure relates to a procedure for identifying a biomarker of a disease or disorder associated with epigenetic modifications based on the quantification of one or more nucleosome modifications, the procedure comprising:

to. isolate a biological sample from the subject;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at one or more target epitopes;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying one or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding one or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard;

g. quantify the abundance of nucleosome modification on target epitopes by comparing the relative abundance in the native nucleosome library with the reference standard; and

h. correlating the abundance of one or more target epitopes with the disease or disorder associated with epigenetic modifications; thus identifying a biomarker of the disease or disorder associated with epigenetic modifications.

A further aspect of the present disclosure relates to a method of selecting an agent that modifies the epigenetic state of one or more nucleosome modifications from a biological sample of a subject, the method comprising determining the quantification of one or more modifications. of nucleosome in the presence and absence of the agent, wherein determining the quantification of one or more nucleosome modifications comprises:

to. isolate a biological sample from the subject;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at one or more target epitopes;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying one or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding one or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard;

g. quantify the abundance of nucleosome modification on target epitopes by comparing the relative abundance in the native nucleosome library with the reference standard;

wherein a change in epigenetic state in the presence and absence of the agent identifies an agent that modifies the epigenetic state.

Brief description of the figures

Figures 1A-1C show the synthesis and quality validation of modified recombinant designer nucleosomes (dNucs). (A) Mass spectrometry of tricitrullinated histone H3R2/R8/R17 before dNuc assembly. Expected mass = 15227 Daltons; actual mass = 15226 Daltons. (B) Validation of octamer and PTM using Coomassie and immunoblotting (Abcam: ab5103) after reducing SDS-PAGE. (C) Validation of dNuc H3R2cit/R8cit/R17cit assembly using native PAGE (no free DNA).

Figures 2A-2B show that nucleosomes are recovered to expected values and provide a reliable calibration in human plasma. A sandwich ELISA was developed to measure nucleosome citrullination, using dNucs and histones H3R2cit/R8cit/R17cit as substrates. Substrates were captured using an anti-H3R2cit/R8cit/R17cit antibody; Detection was performed using an HRP-conjugated antibody specific for the unmodified C terminus of H3. (A) Standard curve using citrullinated dNucs or H3 added to 5% human plasma. (B) Recovery of dNuc or citrullinated H3 added to 100% plasma. Samples were diluted 20-fold before ELISA and normalized to the standards in (A). Theoretical = expected normalized value.

Figures 3A-3C show ELISA optimization for nucleosome PTMs using combinatorially modified dNucs. A capture antibody against a specific PTM (H3R8cit) was paired with a variety of detection antibodies, including (A) an anti-H3 C-terminal antibody, (B) an anti-H3R2cit antibody, and (C) the same anti-H3cit antibody. -H3R8cit used for capture. These capture-detection pairs were tested against a combinatorially modified dNuc (H3R2cit/R8cit/R17cit) and unmodified dNucs in ELISA to identify reagents with low background and high specificity.

Figures 4A-4B show that recombinant modified dNucs are recovered and reliably quantified from human plasma. The optimized sandwich ELISA uses an anti-H3R8cit antibody for substrate capture, followed by incubation with a biotinylated anti-H3R2cit antibody and detection with streptavidin-HRP. (A) dNucs H3R2cit/R8cit/R17cit were added to standard ELISA buffer to generate a standard curve. (B) dNucs H3R2cit/R8cit/R17cit were added to varying dilutions of healthy human plasma and the percent recovery was interpolated from the standard curve shown in (A).

Figures 5A-5D show estimated concentrations of dNucs from endogenous circulating modified nucleosomes in human plasma. (A, C) dNucs H3R2cit/R8cit/R17cit were used to generate standard curves to quantify nucleosome citrullination using linear (A) and nonlinear (C) regression procedures. (B, D) Plasma from patients diagnosed with severe and moderate rheumatoid arthritis (RA), a pathology associated with high levels of nucleosome citrullination in blood, and healthy controls were diluted and analyzed by ELISA. (B) Linear dilution of human plasma samples parallels the linear standard curve (A), establishing dNucs as suitable references for endogenous nucleosomes. (D) Normalization using nonlinear standard curves provides accurate and consistent recovery over a wide range of plasma dilutions and shows altered levels of nucleosome citrullination between controls and moderate to severe RA. For all images in Figures 5A-5D, an ELISA was performed using an anti-H3R8cit antibody for substrate capture, followed by incubation with biotinylated anti-H3R2cit antibody and detection with streptavidin-HRP.

Detailed description of the invention

The invention is defined by the appended claims included herewith. This disclosure is explained in more detail below. For example, features illustrated with respect to one embodiment of the disclosure may be incorporated into other embodiments of the disclosure, and features illustrated with respect to a particular embodiment of the disclosure may be deleted from that embodiment of the disclosure. disclosure. Furthermore, in light of the present disclosure, numerous variations and additions to the various embodiments of the disclosure suggested herein will be apparent to those skilled in the art that do not depart from the present disclosure. Therefore, the following specification is intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations and variations thereof.

Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein may be used in any combination. Furthermore, the present disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein may be excluded or omitted. By way of illustration, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, may be omitted individually or in any combination.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present disclosure pertains. The terminology used in the description of the disclosure herein is solely for the purpose of describing particular embodiments of the disclosure and is not intended to be limiting of the disclosure.

Nucleotide sequences are presented herein by means of only a single strand, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature, or (for amino acids) by the one-letter code or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established use.

Unless otherwise indicated, standard procedures known to those skilled in the art may be used for the production of recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, the manipulation of nucleic acid sequences, the production of transformed cells, the construction of nucleosomes and transiently and stably transfected cells. These techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, New York, 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

As used in the description of the disclosure, the singular forms "a", "an", "the" and "the" are intended to also include the plural forms, unless the context clearly indicates otherwise.

As used herein, "and/or" refers to, and encompasses, any and all possible combinations of one or more of the associated enumerated elements, as well as the lack of combinations when interpreted as alternative ("or").

Furthermore, the present disclosure also contemplates that in some embodiments of the disclosure, any feature or combination of features set forth herein may be excluded or omitted.

Furthermore, the term "about", as used herein when referring to a measurable value such as an amount of a compound or agent of the present disclosure, dose, time, temperature and the like, is intended to encompass variations of ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified amount.

The term "consisting essentially of", as used herein with respect to a nucleic acid or a protein, means that the nucleic acid or protein does not contain any element other than the mentioned element(s) that significantly alters ( for example, more than about 1%, 5% or 10%) the function of interest of the nucleic acid or protein.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description that refers to a polypeptide applies equally to a description of a peptide and to a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are a non-natural amino acid. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide and/or pseudopeptide bonds.

A "nucleic acid" or "nucleotide sequence" is a sequence of nucleotide bases, and may be sequences of RNA, DNA or DNA-RNA hybrids (including nucleotides of both natural and non-natural origin), but preferably are single-stranded or double-stranded DNA sequences.

As used herein, an "isolated" nucleic acid or nucleotide sequence (e.g., an "isolated DNA" or an "isolated RNA") means a nucleic acid or nucleotide sequence separated from or substantially devoid of at least some of the other components of the organism or naturally occurring viruses, for example, the cell or viral structural components or other polypeptides or nucleic acids that are commonly found associated with the nucleic acid or nucleotide sequence.

Likewise, an "isolated" polypeptide means a polypeptide that has been separated from or is substantially devoid of at least some of the other components of the naturally occurring organism or virus, for example, cellular or viral structural components or other polypeptides or nucleic acids that They are commonly found associated with the polypeptide.

By "substantially preserving" a property, it means that at least approximately 75%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the property (for example, activity or other measurable characteristic).

The term "epitope" refers to any site on a biomolecule that can trigger the binding of an affinity reagent. The affinity reagent could recognize a linear sequence of a biomolecule or biomolecule fragment, the shape of a biomolecule or biomolecule fragment, a chemophysical property of a biomolecule or biomolecule fragment, or a combination thereof.

"Amino acids" may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Amino acid residues in proteins or peptides are abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Being or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine is Cys or C; tryptophan is Trp or W; arginine is Arg or R; and glycine is Gly or G.

The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that operate in a manner similar to naturally occurring amino acids. The naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is, a carbon that is bonded to a hydrogen, a carboxyl group, an amino group and an R group, such as homoserine, norleucine, methionine sulfoxide and methionine-methyl-sulfonium. Such analogues have modified R groups (such as norleucine) or modified peptide chains, but retain the same basic chemical structure as a naturally occurring amino acid.

Regarding amino acid sequences, one skilled in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete a single amino acid or a small percentage of amino acids in The encoded sequence is a "conservatively modified variant" in which the alteration results in the substitution of one amino acid for a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are known to those skilled in the art. Such conservatively modified variants are additional and do not exclude polymorphic variants, interspecies homologs/orthologs and alleles of the agents described herein.

An "antigen", as used herein, can be any structure that is recognized by an antibody or for which recognition antibodies (or analogous affinity reagents such as aptamers or selected phages) can be generated. In certain embodiments of the disclosure, the antigens may comprise a single amino acid residue or an amino acid fragment of 2 or more residues. In certain embodiments of the disclosure, the antigens may comprise modifications of an amino acid, such as acetylation, methylation (e.g., mono-, di-, tri-, symmetrical, asymmetrical), phosphorylation, ubiquitination (e.g., mono- , di-, tri-, poly-), sumoylation, ADP-ribosylation, citrullination, biotinylation and cis-trans isomerization. In certain embodiments of the disclosure, the antigens may comprise nucleotide modifications, such as 5-methylcytosine. In other embodiments of the disclosure, the antigens may comprise specific mutations, such as point mutations. In still other embodiments of the disclosure, the antigens may comprise wild-type amino acid sequences or nucleotide sequences.

The term "post-translational modification" refers to any modification of a natural or non-natural amino acid that occurs or would occur in such amino acid after it has been incorporated into a polypeptide chain in vivo or in vitro. Such modifications include, but are not limited to, acylation (e.g., acetyl-, butyryl-, crotonyl-), methylation (e.g., mono-, di-, tri-), phosphorylation, ubiquitination (e.g., mono-, di-, tri-, poly-), sumoylation, ADP-ribosylation, citrullination, biotinylation and cis-trans isomerization. These modifications may be introduced synthetically, for example chemically, during polypeptide synthesis or enzymatically after polypeptide synthesis or polypeptide purification.

The term "post-transcriptional modification" refers to any modification of a natural or non-natural nucleotide that occurs or would occur in such nucleotide after it has been incorporated into a polynucleotide chain in vivo or in vitro. Such modifications include, but are not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, 5,6-dihydrouracil, 7-methylguanosine, xanthosine and inosine.

The present invention relates to the use of recombinant/semi-synthetic nucleosomes carrying histone and/or DNA modifications as standards for assay quantification. Unlike the purified chromatin extracts currently used, the present assay uses fully defined recombinant/semi-synthetic nucleosomes for precise quantification of individual or combinatorial nucleosome modifications. The methods of the invention are advantageously capable of providing absolute quantification of histone and/or DNA modifications, that is, the detection of the number of modified molecules instead of, or in addition to, the relative abundance. Similar to standards commonly used in the art, the modified nucleosomes will be assayed in the same experiment (treated identically to the samples) to generate a standard curve for assay quantification. Quantification of individual or combinatorial nucleosome PTMs may provide better disease biomarkers than the relative measurements currently in use. In some embodiments of the disclosure, recombinant nucleosomes can be used as controls to confirm that combinatorial modifications are detected in an individual nucleosome or in adjacent nucleosomes.

As used herein, a recombinant nucleosome (also called a designer nucleosome (dNuc)) is one that has been prepared by bringing together histones (including the core histones H2A, H2B, H3 and H4, and optionally the linker histone H >1<), DNA, and optionally other factors to form the nucleosome. In other words, a recombinant nucleosome is one that is synthesized, not isolated from cells or chromatin. Each histone in the nucleosome can independently be fully synthetic, semisynthetic (e.g., produced recombinantly and linked to a synthetic peptide), or produced recombinantly. Each histone in the nucleosome can be a histone variant (for example, H3.3, H2A.Bbd, H2A.Z.1, H2A.Z.2, H2A.X, mH2A1.1, mH2A1.2, mH2A2 or TH2B) . The term recombinant nucleosome covers semisynthetic nucleosomes and synthetic nucleosomes.

The recombinant nucleosomes of the present disclosure can be used as addition standards in any known chromatin-based immunoassay (or based on an analogous affinity reagent).

Therefore, one aspect of the present disclosure relates to a method for quantifying the abundance of a nucleosome modification (histone or DNA) in a biological sample, the method comprising:

to. isolate a biological sample;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at a target epitope;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising a core histone and/or DNA modification target epitope;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding an affinity reagent to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard; and

g. Quantify the abundance of nucleosome modification at the target epitope by comparing the relative abundance in the native nucleosome library with the reference standard.

Another aspect of the present disclosure relates to a method for quantifying the abundance of two or more modifications (histone or DNA) in an individual nucleosome in a biological sample, the method comprising:

to. isolate a biological sample;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising two or more core histone and/or DNA modifications at a target epitope;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying two or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding two or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard; and

g. Quantify the abundance of nucleosome modification on target epitopes by comparing the relative abundance in the native nucleosome library with the reference standard.

A further aspect of the present disclosure relates to a method for quantifying the abundance of one or more nucleosome modifications (histone or DNA) in a biological sample from a subject suffering from a disease or disorder, the method comprising:

to. isolate a biological sample from the subject;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at one or more target epitopes;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying one or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding one or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard; and

g. Quantify the abundance of nucleosome modification at the target epitope by comparing the relative abundance in the native nucleosome library with the reference standard.

In each of the methods of the disclosure, the biological sample can be any sample from which nucleosomes can be isolated. The biological sample may be, for example, blood, serum, plasma, urine, saliva, semen, prostatic fluid, nipple aspirate, tear fluid, perspiration, feces, cheek swabs, cerebrospinal fluid, cell lysate samples, amniotic fluid, gastrointestinal fluid, biopsy tissue, lymphatic fluid, or cerebrospinal fluid. In some embodiments of the disclosure, the biological sample comprises cells and chromatin is isolated from the cells. In some embodiments of the disclosure, the cells are cells of a disease or disorder associated with changes in one or more post-translational histone modifications and/or DNA modifications, for example, a diseased cell. In some embodiments of the disclosure, the cells are cells of a tissue or organ affected by a disease or disorder associated with histone mutations, for example, a diseased tissue or organ. Cells can be obtained from the diseased organ or tissue by any means known in the art, including, but not limited to, aspiration.

In other embodiments of the disclosure, the cells are not cells of a tissue or organ affected by a disease or disorder associated with changes in histone post-translational modifications or DNA modifications or associated with histone mutations. The cells may be, for example, cells that serve as substitutes for diseased cells. The cells may be cells that are more easily accessible than diseased cells, for example, which can be obtained without the need for complicated or painful procedures such as biopsies. Examples of suitable cells include, without limitation, peripheral blood mononuclear cells.

In some embodiments of the disclosure, the biological sample is a biopsy. In other embodiments of the disclosure, the biological sample is a biological fluid. In some embodiments of the disclosure, the biological sample comprises peripheral blood mononuclear cells. In other embodiments of the disclosure, the biological sample comprises circulating nucleosomes, for example, released from dying cells. Circulating nucleosomes can be, for example, from blood or cells from a disease or disorder associated with epigenetic modifications. In certain embodiments of the disclosure, the biological sample is plasma, urine, saliva, feces, lymphatic fluid, or cerebrospinal fluid. In some embodiments of the disclosure, the biological sample may be treated with an enzyme to digest chromatin into mononucleosomes and/or polynucleosomes. The enzyme may be, without limitation, a nuclease, for example, micrococcal nuclease.

The subject may be any subject for whom the procedures of the present disclosure are desired. In some embodiments of the disclosure, the subject is a mammal, for example, a human. In some embodiments of the disclosure, the subject is a laboratory animal, for example, a mouse, rat, dog or monkey, for example, an animal model of a disease. In certain embodiments of the disclosure, the subject may be someone who has been diagnosed with or is suspected of having a disease or disorder. In some embodiments of the disclosure, the subject may be someone who is at risk of developing a disease or disorder, for example, due to genetics, family history, exposure to toxins, etc.

The affinity agent used in the methods of the disclosure can be any agent that recognizes and specifically binds to a histone or DNA modification of interest present on a target epitope. In some embodiments of the disclosure, the affinity agent is an antibody or antibody fragment directed toward the epitope. The antibody or fragment thereof may be a full-length immunoglobulin molecule, a <Fab, a Fab', an F(ab)'>2<, a scFv, an Fv fragment, a nanobody, a VHH or a minimal unit recognition.>The affinity agent may be an aptamer or a non-immunoglobulin structure, such as an afibody, an aphylin molecule, an adnectin, a lipocalin mutein, a DARPin, a knottin, a Kunitz-like domain , an avimer, a tetranectin or a trans-body.

In some embodiments of the disclosure, quantification of at least one or more nucleosome modifications is determined by an antibody-based detection assay. Examples of antibody-based detection methods include, without limitation, ChIP, ELISA, AlphaLISA, AlphaSCREEN, Luminex, and immunoblotting. In some embodiments of the disclosure, the antibody-based detection assay uses two different antibodies for capture and detection of the substrate. In one embodiment of the disclosure, the capture and detection antibodies can specifically bind to a histone PTM and another nucleosome structure, such as DNA or unmodified histone, respectively (as shown in Figure 3A). In another embodiment of the disclosure, the capture and detection antibodies are for two different histone PTMs, respectively (as shown in Figure 3B). In some embodiments of the disclosure, the antibody-based detection assay uses the same antibody for both capture and detection of the substrate (as shown in Figure 3C).

Histone or DNA modification that can be quantified by the methods of the disclosure includes any modification that is known in the art or is identified in the future. Known post-translational amino acid modifications include, without limitation, N-acetylation of serine and alanine; phosphorylation of serine, threonine, and tyrosine; N-crotonylation, N-lysine acylation; N>6<-methylation, N>6<,N>6<-dimethylation, N>6<,N>6<,N>6<-lysine trimethylation;>omega-N-methylation, symmetric dimethylation, asymmetric dimethylation of arginine; arginine citrullination; lysine ubiquitinylation; lysine sumoylation; O-methylation of serine and threonine, ADP-ribosylation of arginine, aspartic acid and glutamic acid; and oncogenic mutations (e.g., H3K4M, H3K9M, H3K27M, H3G34R, H3G34V, H3G34W, or H3K36M). Known post-transcriptional DNA modifications include, without limitation, 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine, 3-methylcytosine, 5,6-dihydrouracil, 7-methylguanosine, xanthosine and inosine.

The target epitope may be any core histone or DNA epitope for which quantification and/or monitoring is desired. In some embodiments of the disclosure, the epitope is a post-translational modification or protein isoform. In some embodiments of the disclosure, the core histone epitope comprises at least one post-translational amino acid modification, for example, selected from the group consisting of N-acetylation of serine and alanine; serine, threonine and tyrosine phosphorylation; N-acylation of lysine (for example, crotonylation or butyrylation); N>6<-methylation, N>6<,N>6<-dimethylation, N>6<,N>6<,N>6<-trimethylation of lysine; omega-N->methylation, symmetric dimethylation, asymmetric dimethylation of arginine; arginine citrullination; lysine ubiquitinylation; lysine sumoylation; O-methylation of serine and threonine; serine, threonine or tyrosine phosphorylation; ADP-ribosylation of arginine, aspartic acid and glutamic acid, and any combination thereof. Modification<can be any of those listed in the table>1<(a>)-1<(f), either alone or in any combination.>

In some embodiments of the disclosure, the epitope is a mutation in a core histone, for example, a mutation associated with a disease or disorder. In some embodiments of the disclosure, the mutation is an oncogenic mutation, for example, a mutation that includes, but is not limited to, H3K4M, H3K9M, H3K27M, H3G34R, H3G34V, H3G34W, H3K36M and any combination thereof. H3 mutants can be based on any H3 variant backbone, for example, H3.1, H3.2 or H3.3.

A further aspect of the present disclosure relates to a method for determining a prognosis for a subject suffering from a disease or disorder associated with epigenetic modifications based on the quantification of one or more nucleosome modifications, the method comprising:

to. isolate a biological sample from the subject;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at one or more target epitopes;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying one or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding one or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard;

g. quantify the abundance of nucleosome modification on target epitopes by comparing the relative abundance in the native nucleosome library with the reference standard; and

h. determine the prognosis of the subject based on the abundance of one or more target epitopes.

The details described above for the procedure for detecting and quantifying the presence of nucleosome modifications also apply to this procedure.

In some cases, the epigenetic status of an epitope is indicative of the prognosis of a disease or disorder associated with epigenetic modifications. Therefore, a determination of the epigenetic status of an epitope in a subject who has been diagnosed or suspected of having a disease or disorder associated with epigenetic modifications may be useful in determining the prognosis for the subject. Many of these examples are known in the art. An example is prostate cancer and histone PTMs, which include, without limitation, increased H3K18 acetylation and H3K4 dimethylation associated with a significantly increased risk of prostate tumor recurrence, H4K12 acetylation and dimethylation of H4R3 correlated with tumor stage, and H3K9 dimethylation associated with low-grade prostate cancer patients at risk of recurrence. Another example is the link between overall survival in breast cancer patients and the methylation status of the CpGs in the CREB5, EXPH5, ZNF775, ADCY3 and ADMA genes>8. Another example is glioblastoma and hypermethylation of intronic regions of genes such as EGFR, P<t>E<n>, NF1, PIK3R1, RB1, PDGFRA and QKI. Another example is the inferior prognosis for colon cancer and the promoter methylation status of the CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 genes.

Another aspect of the present disclosure relates to a procedure for identifying a biomarker of a disease or disorder associated with epigenetic modifications based on the quantification of one or more nucleosome modifications, the procedure comprising:

to. isolate a biological sample from the subject;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at one or more target epitopes;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying one or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding one or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard;

g. quantify the abundance of nucleosome modification on target epitopes by comparing the relative abundance in the native nucleosome library with the reference standard; and

h. correlating the abundance of one or more target epitopes with the disease or disorder associated with epigenetic modifications; thus identifying a biomarker of the disease or disorder associated with epigenetic modifications.

The details described above for the procedure for detecting and quantifying the presence of a nucleosome modification also apply to this procedure.

In this procedure, biological samples of diseased tissue can be taken from various patients suffering from a disease or disorder and the epigenetic status of one or more epitopes can be determined. Correlations between epitope status and incidence, stage, subtype, prognosis, etc. can then be identified using analytical techniques that are well known in the art.

A further aspect of the present disclosure relates to a method of selecting an agent that modifies the epigenetic state of one or more nucleosome modifications from a biological sample of a subject, the method comprising determining the quantification of one or more modifications. of nucleosome in the presence and absence of the agent, wherein determining the quantification of one or more nucleosome modifications comprises:

to. isolate a biological sample from the subject;

b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at one or more target epitopes;

c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising nucleosomes carrying one or more histone and/or DNA modification target epitopes;

d. providing the recombinant nucleosome sample at various concentrations to create a reference standard;

and. adding one or more affinity reagents to the native nucleosome library and recombinant nucleosome references;

F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard;

g. quantify the abundance of nucleosome modification on target epitopes by comparing the relative abundance in the native nucleosome library with the reference standard;

wherein a change in epigenetic state in the presence and absence of the agent identifies an agent that modifies the epigenetic state.

The details described above for the procedure of detecting and quantifying the presence of a nucleosome modification also apply to this procedure.

The selection procedure can be used to identify agents that increase or decrease epigenetic modifications. In some embodiments of the disclosure, the detected increase or decrease is statistically significant, for example, at least p < 0.05, for example, p < 0.01, 0.005 or 0.001. In other embodiments of the disclosure, the detected increase or decrease is at least approximately >10%, >20%, >30%, 40%, 50%, 60%, 70%, 80%, 90 %, 100% or higher.

According to the present disclosure, any compound of interest can be selected. Suitable test compounds include organic and inorganic molecules. Suitable organic molecules may include, but are not limited to, small molecules (compounds less than about 1000 Daltons), polypeptides (including enzymes, antibodies and antibody fragments), carbohydrates, lipids, coenzymes and nucleic acid molecules (including DNA, RNA and chimeras and analogues thereof) and nucleotides and nucleotide analogues.

Additionally, the methods of the disclosure can be practiced to screen a library of potential or specific regulators, for example, a small molecule library, a combinatorial chemical compound library, a polypeptide library, a cDNA library, a of antisense nucleic acids, an siRNA library and the like, or a collection of compounds on matrices such as polypeptide and nucleic acid matrices.

Any suitable selection assay format may be used, for example, high-throughput selection.

The procedure can also be used to characterize agents that have been identified as agents that modify the epigenetic state of a specific genomic locus on chromatin. Characterization, for example, preclinical characterization, may include, for example, determining effective concentrations, determining effective dosing schedules, and measuring pharmacokinetics and pharmacodynamics.

A further aspect of the disclosure relates to a method for monitoring changes in epigenetic state over time in the chromatin of a biological sample from a subject, using the methods of the disclosure.

The details described above for the procedure of detecting and quantifying the presence of a nucleosome modification also apply to this procedure.

The steps of the procedure can be repeated as many times as desired to monitor changes in the state of an epigenetic modification, for example, 2, 3, 4, 5,>6<, 7,>8<, 9, 10, 25 , 50 or 100 or more times. The procedure can>be repeated according to a regular schedule (for example, daily, weekly, monthly, yearly) or on an as-needed basis. The procedure may be repeated, for example, before, during and/or after therapeutic treatment of a subject; after a diagnosis of a disease or disorder in a subject; as part of determining a diagnosis of a disease or disorder in a subject; after the identification of a subject who is at risk of developing a disease or disorder, or any other situation in which it is desirable to monitor possible changes in epigenetic modifications or mutations.

A further aspect of the disclosure relates to a method for monitoring the effectiveness of an epigenetic therapy in a subject suffering from a disease or disorder associated with epigenetic modifications, the method comprising monitoring changes in the epigenetic state over time in the chromatin. of a biological sample from the subject, using the procedures of the disclosure.

The details described above for the procedure for detecting and quantifying the presence of a nucleosome modification also apply to this procedure.

Epigenetic therapies are those designed to alter the epigenetic state of proteins (e.g., histones) or DNA. An example of epigenetic therapy includes lysine deacetylase inhibitors (also called histone deacetylase inhibitors) (for example, vorinostat (suberoylanilide-hydroxamic acid), CI-994 (tacedinaline), MS-275 (entinostat), BMP-210, M344, n V p -La Q824, LBH-529 (panobinostat), MGCD0103 (mocetinostat), PXD101 (belinostat), CBHA, PCI-24781, ITF2357, valproic acid, trichostatin A and sodium butyrate), which are used to treat lymphoma T-cell therapy (CTCL) or in clinical trials for the treatment of hematologic and solid tumors, including lung, breast, pancreatic, kidney, and bladder cancers, melanoma, glioblastoma, leukemias, lymphomas, and multiple myeloma. Another example of epigenetic therapy is lysine acetyltransferase inhibitors (also called histone acetyltransferase inhibitors) (e.g., epigallocatechin-3-gallate, garcinol, anacardic acid, CPTH2, curcumin, MB-3, MG149, C646, and romidepsin). Another example of epigenetic therapy is DNA methyltransferase inhibitors (e.g., azacytidine, decitabine, zebularine, caffeic acid, chlorogenic acid, epigallocatechin, hydralazine, procainamide, procaine, and RG108), which have been approved for the treatment of acute myeloid leukemia. , myelodysplastic syndrome and chronic myelomonocytic leukemia and in clinical trials for the treatment of solid tumors. Other epigenetic therapies include, without limitation, lysine methyltransferases (e.g., pinomethostat, tazomethostat, CPI-1205); lysine demethylases (eg, ORY1001); arginine methyltransferases (eg, EPZ020411); arginine deiminases (eg, GSK484); and isocitrate dehydrogenases (e.g., enasidenib, ivosidenib). See Fischle et al., ACS Chem. Biol. 11:689 (2016); DeWoskin et al., Nature Rev. 12:661 (2013); Campbell et al., J. Clin. Invest. 124:64 (2014); and Brown et al., Future Med. Chem. 7:1901 (2015).

The steps of the procedure can be repeated as many times as desired to monitor the effectiveness of the treatment, <for example, 2, 3, 4, 5,>6<, 7,>8<, 9, 10, 25, 50 or 100 or more times. The procedure may be repeated on a regular schedule (e.g. daily, weekly, monthly, yearly) or on an as-needed basis, e.g. until completion of therapeutic treatment. The procedure may be repeated, for example, before, during and/or after therapeutic treatment of a subject, for example, after each administration of the treatment. In some embodiments of the disclosure, the treatment continues until the procedure of the disclosure shows that the treatment has been effective.

Another aspect of the disclosure relates to a procedure for selecting a suitable treatment for a subject suffering from a disease or disorder associated with epigenetic modifications based on the epigenetic state in chromatin from a biological sample of the subject using the methods of the disclosure. .

The details described above for the procedure for detecting and quantifying the presence of a nucleosome modification or mutation also apply to this procedure.

The procedure can be applied, for example, to subjects who have been diagnosed or suspected of suffering from a disease or disorder associated with epigenetic modifications. A determination of the epigenetic status of an epitope may indicate that the status of an epitope has been modified and an epigenetic therapy should be administered to the subject to correct the modification. In contrast, a determination that the status of an epitope is unchanged would indicate that an epigenetic therapy is not expected to be effective and should be avoided. For example, a determination that a particular acetylated histone residue (H3K27ac) has been deacetylated may indicate that treatment with a lysine deacetylase inhibitor would be appropriate. Similarly, the determination that a particular methylated histone residue (H3K27me3) has become hypermethylated may indicate that treatment with a lysine methyltransferase inhibitor would be appropriate.

As used herein, a disease or disorder associated with epigenetic modifications is any disease or disorder in which an epigenetic modification is known to be the cause of the disease or disorder or at least a symptom of the disease or disorder or a disease or disorder in which an epigenetic modification is a biomarker of the disease or disorder. In any of the methods of the disclosure, the disease or disorder associated with epigenetic modifications or mutations may be a cancer, a central nervous system (CNS) disorder, an autoimmune disorder, an inflammatory disorder or an infectious disease.

Cancer can be any abnormal growth of cells, benign or malignant, including, but not limited to, acoustic neuroma, acute granulocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, adenocarcinoma, adrenal carcinoma, adrenal cortex carcinoma, anal cancer, anaplastic astrocytoma , angiosarcoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, brain cancer, breast cancer, bronchogenic carcinoma, cervical carcinoma, cervical hyperplasia, chordoma, choriocarcinoma, chronic granulocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystaadenosarcoma, embryonal carcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, esophageal carcinoma, essential thrombocytosis, Ewing tumor, fibrosarcoma, genitourinary carcinoma, glioblastoma, glioma, gliosarcoma, cell leukemia hairy, head and neck cancer, hemangioblastoma, liver carcinoma, Hodgkin's disease, Kaposi sarcoma, leiomyosarcoma, leukemia, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoma, malignant carcinoid carcinoma, malignant hypercalcemia, malignant melanoma, malignant pancreatic insulinoma , mastocytoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, mycosis fungoides, myeloma, myxoma, myxosarcoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung carcinoma, oligodendroglioma, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenosarcoma, papillary sarcoma, pinealoma, polycythemia vera, primary brain carcinoma, primary macroglobulinemia, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sebaceous gland sarcoma, seminoma, skin cancer, small cell lung carcinoma, soft tissue sarcoma, squamous cell carcinoma, stomach carcinoma, sweat gland carcinoma, synovioma, testicular carcinoma, throat cancer, thyroid carcinoma and Wilms tumor.

CNS disorders include genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Illustrative diseases of the CNS include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay Sachs disease, Lesch-Nyan disease, epilepsy, stroke, psychiatric disorders, including mood disorders (e.g., depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder, mania, manic psychosis), schizophrenia, schizoaffective disorder, schizophreniform disorder, drug dependence (e.g., alcoholism and other drug dependencies) , neuroses (e.g., anxiety, obsessive disorder, somatoform disorder, dissociative disorder, grief, postpartum depression), psychoses (e.g., hallucinations and delusions, psychosis not otherwise specified (psychosis NOS), dementia, aging, paranoia, disorder attention deficit disorders, psychosexual disorders, sleep disorders, pain disorders, eating or weight disorders (for example, obesity, cachexia, anorexia nervosa and bulemia), ophthalmic disorders affecting the retina, posterior tract and optic nerve (for example, retinitis pigmentosa, diabetic retinopathy and other degenerative diseases of the retina, uveitis, age-related macular degeneration, glaucoma), and cancers and tumors (for example, pituitary tumors) of the CNS.

Autoimmune and inflammatory diseases and disorders include, but are not limited to, myocarditis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, subacute bacterial endocarditis, anti-glomerular basement membrane nephritis, interstitial cystitis, lupus nephritis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, anti-synthetase syndrome. , sinusitis, periodontitis, atherosclerosis, dermatitis, allergy, allergic rhinitis, allergic airway inflammation, chronic obstructive pulmonary disease, eosinophilic pneumonia, eosinophilic esophagitis, hypereosinophilic syndrome, graft versus host disease, atopic dermatitis, tuberculosis, asthma, peptic ulcer chronic, alopecia areata, autoimmune angioedema, autoimmune progesterone dermatitis, autoimmune urticaria, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, discoid lupus erythematosus, epidermolysis bullosa acquisita, erythema nodosum, pemphigoid gestationis, hidradenitis suppurativa, lichen planus, lichen sclerosus, Linear IgA, morphea, pemphigus vulgaris, pityriasis lichenoid and acute varioliformis, Mucha-Habermann disease, psoriasis, systemic scleroderma, vitiligo, Addison's disease, <autoimmune polyendocrine syndrome type>1<, autoimmune polyendocrine syndrome type>2<, polyendocrine syndrome >autoimmune type 3, autoimmune pancreatitis, diabetes mellitus type 1, autoimmune thyroiditis, Ord's thyroiditis, Graves' disease, autoimmune oophoritis, endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmune enteropathy, celiac disease, Crohn's disease, irritable bowel syndrome , diverticulitis, microscopic colitis, ulcerative colitis, antiphospholipid syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome, autoimmune neutropenia, autoimmune thrombocytopenic purpura, cold agglutinin disease, essential mixed cryoglobulinemia, Evans syndrome, pernicious anemia, pure erythrocyte aplasia, thrombocytopenia, painful adiposis, adult Still's disease, ankylosing spondylitis, CREST syndrome, drug-induced lupus, enthesitis-related arthritis, eosinophilic fasciitis, Felty syndrome, IgG4-related disease, juvenile arthritis, Lyme disease (chronic), disease mixed connective tissue, palindromic rheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, psoriatic arthritis, reactive arthritis, relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schnitzler syndrome, systemic lupus erythematosus, undifferentiated connective tissue, dermatomyositis, fibromyalgia, myositis, myasthenia gravis, neuromyotonia, paraneoplastic cerebellar degeneration, polymyositis, acute disseminated encephalomyelitis, acute motor axonal neuropathy, anti-N-methyl-D-aspartate receptor encephalitis, Balo concentric sclerosis, Bickerstaff encephalitis , chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, Hashimoto encephalopathy, idiopathic inflammatory demyelinating diseases, Lambert-Eaton myasthenic syndrome, multiple sclerosis, Oshtoran syndrome, pediatric autoimmune neuropsychiatric disorder associated with streptococci (PANDAS), progressive inflammatory neuropathy, restless legs syndrome, stiff person syndrome, Sydenham's chorea, transverse myelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome, Graves ophthalmopathy, intermediate uveitis, woody conjunctivitis, Mooren's ulcer, neuromyelitis optica, opsoclonus myoclonus syndrome, optic neuritis , scleritis, Susac syndrome, sympathetic ophthalmia, Tolosa-Hunt syndrome, autoimmune inner ear disease, Ménière's disease, Behcet's disease, eosinophilic granulomatosis with polyangiitis, giant cell arteritis, granulomatosis with polyangiitis, IgA vasculitis, Kawasaki, leukocytoclastic vasculitis, lupus vasculitis, rheumatoid vasculitis, microscopic polyangiitis, polyarteritis nodosa, polymyalgia rheumatica, urticaria vasculitis, vasculitis and primary immunodeficiency.

The term "infectious diseases", as used herein, refers to any disease associated with infection by an infectious agent. Examples of infectious agents include, but are not limited to, viruses and microorganisms (e.g., bacteria, parasites, protozoa, cryptosporidia). Viruses include, without limitation, Hepadnaviridae, including hepatitis A, B, C, D, E, F, G, etc.; Flaviviridae, including human hepatitis C virus (HCV), yellow fever virus, and dengue viruses; Retroviridae, including human immunodeficiency virus (HIV) and human T lymphotropic viruses (HTLV1 and HTLV2); Herpesviridae, including herpes simplex viruses (HSV-1 and HSV-2), Epstein Barr virus (EBV), cytomegalovirus, varicella-zoster virus (VZV), human herpesvirus 6 (HHV ->6<) and human herpesvirus>8<(HHV->8<) and herpesvirus B; Papovaviridae, including human papillomavirus; Rhabdoviridae, including rabies virus; Paramyxoviridae, including respiratory syncytial virus; Reoviridae, including rotavirus; Bunyaviridae, including hantaviruses; Filoviridae, including Ebola virus; Adenoviridae; Parvoviridae, including parvovirus B-19; Arenaviridae, including Lassa virus; Orthomyxoviridae, including influenza viruses; Poxviridae, including Orf virus, molluscum contagiosum virus, smallpox virus, and monkeypox virus; Togaviridae, including Venezuelan equine encephalitis virus; Coronaviridae, including coronaviruses such as severe acute respiratory syndrome (SARS) virus; and Picornaviridae, including polioviruses; rhinovirus; orbivirus; picodnavirus; encephalomyocarditis virus (EMV); parainfluenza virus, adenovirus, Coxsackie virus, Echo virus, rubella virus, rubella virus, human papillomavirus, canine distemper virus, contagious canine hepatitis virus, feline calicivirus, feline rhinotracheitis virus, virus GET (swine), foot and mouth disease virus, simian virus 5, human parainfluenza virus type 2, human metapneumomovirus, enterovirus and any other pathogenic virus currently known or subsequently identified (see, for example, Fundamental Virology, Fields et al. al., Eds., 3rd ed., Lippincott-Raven, New York, 1996).

Pathogenic microorganisms include, but are not limited to, Rickettsia, Chlamydia, Chlamydophila, Mycobacteria, Clostridia, Corynebacteria, Mycoplasma, Ureaplasma, Legionella, Shigella, Salmonella, pathogenic Escherichia coli species, Bordatella, Neisseria, Treponema, Bacillus, Haemophilus, Moraxella, Vibrio , Staphylococcus spp., Streptococcus spp., Campylobacter spp., Borrelia spp., Leptospira spp., Erlichia spp., Klebsiella spp., Pseudomonas spp., Helicobacter spp. and any other pathogenic microorganism currently known or subsequently identified (see, for example, Microbiology, Davis et al, Eds., 4th ed., Lippincott, New York, 1990). Specific examples of microorganisms include, but are not limited to, Helicobacterpylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Treponema pal lidum, Bacillus anthracis, Salmonella typhi, Vibrio cholera, Pasteurella pestis (Yersinia pestis), Pseudomonas aeruginosa, Campylobacter jejuni, Clostridium difficile, Clostridium botulinum, Mycobacterium tuberculosis, Borrelia burgdorferi, Haemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, emophilus influenza , Listeria monocytogenes, Shigella flexneri, Anaplasma phagocytophilum, enterotoxic Escherichia coli and Schistosoma haematobium.

Another aspect of the disclosure relates to methods for determining the specificity of a detection reagent (e.g., an antibody or fragment thereof, an aptamer, etc.) for a core histone and/or DNA modification target epitope, comprising using the recombinant mononucleosome and/or polynucleosome sample of the disclosure that comprises a core histone and/or DNA modification target epitope.

In some embodiments of the disclosure, the disease or disorder includes, but is not limited to, obesity, diabetes, heart disease, autism, fragile Beckwith Wiedemann, Rett syndrome, Rubinstein-Taybi syndrome, Coffin-Lowry syndrome, immunodeficiency syndrome-centrometric instability-facial anomalies, a-thalassemia, leukemia, Cornelia de Langue syndrome, Kabuki syndrome, progressive systemic sclerosis and hypertrophy cardiac.

In some embodiments of the disclosure, the disease or disorder associated with epigenetic modifications is selected from the group consisting of renal cell carcinoma, glioma, gliosarcoma, anaplastic astrocytoma, medulloblastoma, lung cancer, small cell lung carcinoma, cervical, colon cancer, rectal cancer, chordoma, throat cancer, Kaposi sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, colorectal cancer, endometrial cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, cell carcinoma renal, liver carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, testicular tumor, Wilms tumor, Ewing tumor, bladder carcinoma, angiosarcoma, endotheliosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland sarcoma, papillary sarcoma, papillary adenosarcoma , cystaadenosarcoma, bronchogenic carcinoma, medullary carcinoma, mastocytoma, mesothelioma, synovioma, melanoma, leiomyosarcoma, rhabdomyosarcoma, neuroblastoma, retinoblastoma, glioblastoma, oligodendroglioma, acoustic neuroma, hemangioblastoma, meningioma, pinealoma, ependymoma, craniopharyngioma, epithelial carcinoma, embryonal carcinoma, carcinoma squamous cell, basal cell carcinoma, fibrosarcoma, myxoma, myxosarcoma, glioma, liposarcoma, infections caused by Helicobacter pylori, Listeria monocytogenes, Shigella flexneri, Anaplasma phagocytophilum, Chlamydophila, Epstein-Barr virus, herpes, HIV, Schistosoma haematobium; obesity, diabetes, heart disease; autism, fragile facial abnormalities, athalassemia, leukemia, Huntington's disease, schizophrenia, bipolar disease, aging, dementia, Alzheimer's disease, Parkinson's disease, Cornelia de Langue syndrome, Kabuki syndrome, Sjogren's syndrome, vitiligo, progressive systemic sclerosis, psoriasis, primary biliary cirrhosis, Crohn's disease and ulcerative colitis, Hashimoto's thyroiditis, Graves' disease, inflammatory bowel disease, atherosclerosis and cardiac hypertrophy.

Another aspect of the disclosure provides reagents and kits that include reagents for carrying out one of the procedures described herein. The reagents may be included in suitable containers or containers. The kit may include one or more reagents containing recombinant nucleosomes as described herein for the quantification of epitopes, for example in antibody-based detection assays. The kit may also include at least one affinity reagent as described herein, for example an antibody or a fragment or variant thereof.

In some embodiments of the disclosure, recombinant nucleosomes include DNA-protein complexes made with histones, histone isoforms, histone post-translational modifications, or histone mutations. In various embodiments of the disclosure, any core histone sequence variant known in the art, or post-translational modification, including those defined in Tables >1<(a>)-1<(f),> can be installed on histones comprising the histone octamer. In one embodiment of the disclosure, a set of recombinant nucleosomes is provided.

In other embodiments of the disclosure, the kit may include one or more wash buffers (e.g., phosphate-buffered saline) and/or other buffers in containers or containers. The kit may also include reagents necessary for measuring the amount of standard or sample captured.

When a kit is supplied, the different components can be packaged in separate containers and mixed immediately before use. Such packaging of the components separately can allow long-term storage without losing the functions of the active components. Kits with materials with instructions can also be supplied. The instructions may be printed on paper or other substrate and/or may be provided as an electronically readable medium.

In some embodiments of the disclosure, the kit may comprise a panel of patterns representing some or all of the different possibilities of a particular class of PTMs, for example, lysine methylation, lysine acylation or arginine methylation. for example, from a single histone or from multiple histones. The panel may include some or all modifications that are considered relevant for one or more diseases. In some embodiments of the disclosure, the kit may comprise a set of patterns that represent most or all of the different possibilities of histone mutations, for example, oncogenic histone mutations, for example, a single histone or multiple histones. The panels can be used to evaluate the specificity of affinity reagents, monitor technical variability, and standardize experiments.

EXAMPLES

Example 1: Synthesis and quality validation of modified recombinant designer nucleosomes (dNucs)

Recombinant dNucs are ideal ELISA standards as they are highly defined and mimic the endogenous antibody target. Figures 1A-1C show representative quality metrics for dNuc assembly. In this example, a tricitrullinated histone H3R2/R8/R17 (H3R2cit/R8cit/R17cit) was generated using a native chemical ligation approach based on published procedures (Chen et al., Chembiochem 15(14): 2071 (2014)). The resulting modified histone was >95% pure by analytical HPLC and was within one Dalton of the mass expected by high-resolution mass spectrometry (Figure 1A). To generate citrullinated dNucs, the unmodified histone H3 subunit was replaced by histones H3R2cit/R8cit/R17cit during octamer assembly. As expected, the resulting dNuc was immunoreactive with an anti-citrullinated histone antibody (H3R2cit/R8cit/R17cit) (Figure 1B; top) and showed equal stoichiometric proportions of histones (Figure 1B; bottom) and no detectable free DNA ( Figure 1C). These data show that the recombinant dNucs are very pure, making them exceptional standards for ELISAs aimed at quantifying histone PTMs.

Example 2: Nucleosomes recover to expected values and provide reliable calibration in human plasma

Recent advances in cell-free nucleosome (CFN) analysis have made non-invasive liquid biopsies targeting chromatin a potential reality (Holdenrieder et al., Int. J. Cancer 95(2): 114 (2001) ;Holdenrieder et al., Ann. NYAcad. Sci. 1137:180 (2008); (8):1544 (2005)). However, existing assays for quantification of nucleosome PTMs generally use modified histone subunits as assay standards. These fail to recapitulate the binding characteristics of native chromatin and can therefore only provide relative measurements when used as controls in the assay. Furthermore, histones are unstable in plasma/serum, limiting their usefulness in liquid biopsies, which are rapidly becoming the new focus of nucleosome PTM assay and biomarker development.

In Figures 2A-2B, the recovery of citrullinated dNucs and spiked histone H3 subunits was compared to healthy human plasma. To this end, a standard sandwich ELISA was developed to measure histone citrullination: an anti-H3R2cit/R8cit/R17cit antibody was used to capture the substrates, followed by detection with an HRP-conjugated antibody specific for the C terminus. unmodified from H3. In Figure 2A, dNucs or histone H3R2cit/R8cit/R17cit were added at a range of 1000-fold (1 nM to 1 pM) in 5% human plasma. Note that the ELISA signal is dramatically reduced when quantified in plasma with citrullinated histone H3 versus citrullinated dNucs. These results demonstrate: 1) linear recovery of citrullinated dNucs added to plasma; and 2) higher sensitivity of detection of citrullinated dNuc in plasma compared to citrullinated H3 (i.e., wider range of analyte detection).

Reliable calibrants are stable in the assay matrix (e.g., plasma or cell lysates). To determine whether the observed loss of histone signal is due to aberrant aggregation or interactions with plasma proteins, dNuc or histone H3R2cit/R8cit/R17cit substrates were added to 100% plasma, then samples were diluted to 5% plasma and quantified H3 citrullination (Figure 2B). Samples were normalized to standards prepared in 5% plasma (Figure 2A). Notably, citrullinated dNucs were easily recovered to the expected values (Figure 2B). However, citrullinated histone H3 was significantly lower (i.e., not detectable) when added to 100% plasma versus 5%. These data demonstrate that only citrullinated dNucs (not histone H3) provide reliable calibration in plasma samples. Furthermore, the results support the development of dNucs as reliable standards for next-generation liquid biopsy assays, enabling accurate quantification of histone PTMs directly from patient plasma.

Example 3: ELISA Optimization for Nucleosome PTMs Using Combinatorially Modified dNucs

Since dNucs are structurally analogous and show similar binding characteristics to endogenous nucleosomes (Shah et al., Mol. Cell 72(1):162 (2018)), it was reasoned that they can be used to design PTM detection assays of highly sensitive and specific histone. In this case, dNuc H3R2cit/R8cit/R17cit was applied to the development of an ELISA for the detection of nucleosome citrullination. dNucs were first used to identify highly specific antibodies with low binding activity to non-target PTMs. The dNucs were then applied as highly purified substrates for ELISA development, using several pairs of capture and detection antibodies. The three examples depicted in Figures 3A-3C used a PTM-specific capture antibody (anti-H3R8cit) combined with three different types of biotinylated detection antibodies: 1) an antibody targeting an unmodified portion of histone H3 (Figure 3A); 2) an antibody that recognizes another concurrent PTM, H3R2cit (Figure 3B); and 3) the same anti-H3R8cit antibody used for dNuc capture (Figure 3C). The ELISA signal was generated using a streptavidin-conjugated HRP (SA-HRP). For all three experiments shown, unmodified dNuc was included as a negative control, to monitor background signal.

Surprisingly, the assay with the highest sensitivity and lowest background used the same anti-H3R8cit antibody for both capture and detection (Figure 3C, bottom). Targeting different PTMs (Figure 3B) had similar specificity, but showed a slightly elevated background in the unmodified Nuc control. These experiments demonstrate the remarkable capabilities of the antibodies to bind multiple PTMs on the same nucleosome (e.g., H3R8Cit/H3R2Cit or H3R8cit/H3R8cit), which may allow quantification of concurrent PTMs in future studies. Notably, there are two copies of each histone subunit (e.g., H2A, H2B, H3, and H4) in a nucleosome. Therefore, it is unclear whether the ELISA-H3R8cit/H3R2cit combination detects these histone PTMs in cis or trans. The development of nucleosomes that carry modifications asymmetrically (i.e., in which only one of the histone subunits is modified) is required to determine these possible modes of action.

Detection with an anti-histone H3 antibody (Figure 3A, bottom) showed substantially reduced sensitivity compared to detection with PTM antibodies. In particular, this type of capture/detection setup is the typical industrial approach for histone PTM ELISA (using histone-based substrates or templates), suggesting that significant gains in assay specificity and sensitivity can be achieved by taking advantage of the nucleosome structure. Indeed, existing assays measuring nucleosome PTMs are developed using purified histone extracts or human plasma with unknown endogenous levels of the target analyte. As demonstrated in Figures 3A-3C, the generation of highly purified dNucs allows the most sensitive and specific antibody pair to be empirically determined for ELISA, providing assays with unparalleled accuracy and precision. Furthermore, the linear recovery of the dNucs signal supports the continued development of dNucs as nucleosome PTM-specific assay calibrators.

Example 4: dNucs are recovered and reliably quantified from human plasma

The usefulness of dNucs as calibrators in clinical trials depends on their ability to be accurately recovered from a biological matrix, such as plasma. In this regard, one of the optimized sandwich ELISAs of Figures 3A-3C was employed, using anti-H3R8cit antibody for substrate capture, followed by detection with biotinylated anti-H3R2cit antibody and streptavidin-HRP. First, a standard curve was generated using dNucs H3R2cit/R8cit/R17cit added at a six-point concentration range in ELISA buffer (Figure 4A). The standard curve demonstrates a consistent linear recovery of dNucs in ELISA buffer, with an R>2<=>0.98. In parallel experiments, dNucs H3R2cit/R8cit/R17cit (50 ng/ml) were added to varying dilutions of healthy human plasma and the percent recovery was interpolated from the standard curve (Figure 4B). These results demonstrate >95% recovery of dNuc signal in 2X-32X diluted plasma, indicating minimal interference with the assay matrix, and suggest that dNucs provide highly stable and reliable quantitative standards over a wide range of dilutions. of plasma.

Example 5: dNucs estimate concentrations of endogenous circulating modified nucleosomes in human plasma

Full application of dNucs as assay calibrators requires that they behave similarly to endogenous nucleosomes (i.e., binding and sensing characteristics). To this end, plasma was collected from patients with moderate and severe rheumatoid arthritis (RA) symptoms (according to the DAS28 score (Prevoo et al., Arthritis Rheum. 38(1):44 (1995)). Patients with RA have high levels of nucleosome citrullination (Pratesi et al., Ann. Rheum. Dis. 73(7):1414 (2014); Dwivedi et al., FASEB J. 28(7):2840 (2014); Sohn et al. , Arthritis Rheumatol.

67(11 ):2877 (2015)), making them an ideal candidate for this proof-of-concept study. Plasma from RA patients (along with age- and sex-matched healthy controls) was diluted from 2X to 128X and analyzed by ELISA (Figures 5B, 5D). For this study, ELISA was used for the detection of nucleosome citrullination, using an anti-H3R8cit antibody for nucleosome capture and a biotinylated anti-H3R2cit antibody and streptavidin-HRP for detection. Standard curves were generated using dNucs H3R2cit/R8cit/R17cit spiked over a six-point concentration range in ELISA buffer, and analyzed using linear (Figure 5A) and nonlinear (Figure 5C) regression procedures. What is shown here (Figures 5B, 5D) are the averages for one patient from each category (healthy, moderate RA, and severe RA) from our ongoing study.

As shown in Figure 5B, dilution of human plasma samples generated a linear curve similar to that of the standard curve in Figure 5A. These data confirm that the standard curve accurately represented the sample dilution curve, validating the use of the calibrator chosen to quantify the endogenous material. ELISA data were also normalized using the nonlinear standard curve (Figures 5C, 5D), which provided consistent recovery over a wide range of plasma dilutions. Furthermore, these analyzes demonstrate altered levels of nucleosome citrullination between controls and moderate-to-severe RA, supporting the implementation of dNucs as standards for quantification in clinically relevant assays.

TABLE 1(a) Post-translational modifications for human histones H2A type 1/2/3, H2A.X, H2A.Z and H2A.V isoform 1/2/3/4/5

TABLE 1b Ostraductional modifications for human histones H2A.J H2B ti o 1

TABLE 1c Ostraductional modifications for human histone H2B ti or 2/3/F-S

TABLE 1d Translational modifications for the human utative histone H2B ti or 2-D/2-C

TABLE 1e Translational modifications for human histone H3.1/H3.1t/H3.2/H3.3/H3.3C

TABLE 1(f) Post-translational modifications for human histone H3-like centromeric protein A and human histone H4

Claims (15)

1. A method for quantifying the abundance of a histone or DNA modification in a nucleosome in a biological sample from a subject, the method comprising: to. isolate a biological sample; b. preparing a library of native mononucleosomes and/or polynucleosomes from the biological sample, the library comprising nucleosomes comprising one or more core histone and/or DNA modifications at a target epitope; c. preparing a sample of recombinant mononucleosomes and/or polynucleosomes comprising a core histone and/or DNA modification target epitope; d. providing the recombinant nucleosome sample at various concentrations to create a reference standard, assaying recombinant mononucleosomes and/or polynucleosomes to generate a standard curve; and. adding an affinity reagent to the native nucleosome library and to recombinant nucleosome reference samples, the affinity reagent targeting a modified histone or modified DNA; F. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the native nucleosome library and the recombinant nucleosome reference standard; and g. quantify the abundance of histone modification or DNA modification at the target epitope by comparing the relative abundance in the native nucleosome library with the reference standard. 2<. The method of claim >1<, which comprises quantifying the abundance of two or more >histone or DNA modifications in an individual nucleosome in a biological sample, the method comprising adding two or more affinity reagents to the library of native nucleosomes and to references of recombinant nucleosomes. 3. The method of any one of claims 1-2, wherein the biological sample is treated with an enzyme to digest chromatin into mononucleosomes and/or polynucleosomes; optionally wherein the enzyme is micrococcal nuclease. 4. The method of any one of claims 1 to 3, wherein the biological sample comprises cells and chromatin is isolated from the cells. 5. The method of any one of claims 1 to 4, wherein the biological sample is a biological fluid. 6<. The method of any one of claims 1 to 4, wherein the biological sample comprises peripheral blood mononuclear cells. 7. The method of any one of claims 1 to 4, wherein the biological sample comprises circulating nucleosomes; optionally wherein the circulating nucleosomes are from blood or cells from a disease or disorder associated with epigenetic modifications. 8<. The method of any one of claims 1 to 7, wherein the affinity agent is an antibody or a fragment thereof directed toward the epitope. <9. The method of any one of claims 1 to>8<, wherein at least one or more>nucleosome modifications are quantified in the same nucleosome. <10. The method of any of claims 1 to>8<, wherein at least one or more>nucleosome modifications in different nucleosomes are quantified. 11. The method of any one of claims 1 to 10, wherein the quantification of at least one or more nucleosome modifications is determined by an antibody-based detection assay, optionally wherein the antibody-based detection method is selected from the group consisting of ELISA, AlphaLISA, AlphaSCREEN, Luminex and immunoblotting. 12. The method of claim 11, wherein the antibody-based detection assay uses two different antibodies for capture and detection of the substrate; optionally, wherein the capture and detection antibodies specifically bind to one histone PTM and another nucleosome structure, such as DNA or unmodified histone, or wherein the capture and detection antibodies specifically bind to two PTMs of different histones. 13. The method of claim 11, wherein the antibody-based detection assay uses the same antibody for both capture and detection of the substrate. 14. The method of any one of claims 1 to 13, wherein the nucleosome comprises at least one post-translational amino acid modification or DNA modification selected from the group consisting of N<acetylation of serine and alanine; serine, threonine and tyrosine phosphorylation; N-crotonylation, N-lysine acylation; N>6<-methylation, N>6<,N>6<-dimethylation, N>6<,N>6<,N>6<-trimethylation of lysine; omega-N-methylation, symmetric dimethylation,>arginine asymmetric dimethylation; arginine citrullination; lysine ubiquitinylation; lysine sumoylation; O-methylation of serine and threonine, ADP-ribosylation of arginine, aspartic acid and glutamic acid; oncogenic K to M mutations (e.g., H3K4M, H3K9M, H3K27M, H3G34R, H3G34<v>, H3G34W or H3K36M); 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine, 3-methylcytosine, 5,6-dihydrouracil, 7-methylguanosine, xanthosine and inosine. 15. The method of any one of claims 1-2, wherein modified recombinant nucleosomes are used as controls to confirm that combinatorial modifications are detected in an individual nucleosome or in adjacent nucleosomes.